The present invention relates to a capillary electrophoresis apparatus, suitable for readily performing a variety of capillary electrophoresis processes, such as zone electrophoresis, isoelectric focusing, and electrokinetic micelle-chromatography.
Electrophoresis is an electrochemical process, which can be used for separating from each other electrically charged and, with certain special techniques, also uncharged particles, present in an electrolytic solution and having a size which ranges from the smallest ions and molecules to colloidal particles. Depending on the electrical charge and other properties thereof, the particles travel at different speeds in an electrical field. There are several ways of classifying electrophoretic process. One classification is based on a carrier or an apparatus used for eliminating convection in a liquid phase, e.g. a paper, a gel, a column, or a capillary.
Capillary electrophoresis is one of the most rapidly advancing applications of analytic chemistry. In the process, a background solution is contained in such a thin tube, a capillary, that the viscous forces of a liquid preclude convection. The inner diameter of a capillary is usually within the range of 0.01 to 1 mm. Electrophoresis is hence carried out in a free solution for eliminating disturbances caused by a carrier. It is also easy to free a capillary of thermal energy evolved by an electric current, thus enabling the use of a high electric field for a more expeditious separation. In addition, capillary electrophoresis can be readily automated.
In capillary electrophoresis, two vessels containing a electrolytic background solution are interconnected by means of a capillary tube which contains the same solution. Each vessel is provided with an electrode. A sample to be investigated is placed in the inlet end of the capillary as a short zone. Generally, in order to supply a sample, the end of a capillary is moved from the background solution vessel to the sample vessel and back. This operation causes disturbances and distortions in the background solution at the end of a capillary and in the sample zone and leads to an impaired accuracy of the method. It is also inevitable that the current be switched off for the duration of moving the capillary from one vessel to the other, which may cause fluctuations in running conditions. The same drawbacks result also from replacing the background solution during the course of a run.
Reactions occurring on the electrodes also change the composition of a solution contained in the background solution vessels and these changes may propagate into the capillary to cause distortion in the parameters of a test series.
Capillary electrophoresis can be performed by using various applications. The most commonly used applications are capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), capillary isotachophoresis (CITP), and micellar electrokinetic capillary chromatography (MECC). Although different in appearance, these applications are controlled by the same electrochemical laws. Various applications are created by applying various initial and boundary conditions to an electrophoresis system.
The commercially available capillary electrophoresis devices usually allow various electrophoresis applications to be performed. However, such devices are hampered by certain drawbacks or structural features, which limit the easy transition from one application to another and also the changing of system parameters during a run.
Several researchers have introduced technical solutions, capable of partially eliminating the above drawbacks. Virtanen, Acta Polytechnica Scandinavica, Chemistry Including Metallurgy Series, No. 123 (1974), pp. 1-67, employed, as early as in the 1960""s, injection technology which allows the injection of a sample while electric current is on Verheggen et al., J. Chromatogr., 452 (1988), pp. 615-622, and Zare et al., U.S. Pat. No. 5,141,621, have also introduced a method for injecting a sample into a capillary electrophoresis apparatus without switching off electric current. However, these methods and apparatus do not provide means for exploiting the multitude of possibilities offered by the theoretical similarity of various electrophoresis applications.
An object of the present invention is to provide a novel capillary electrophoresis apparatus, which is capable of eliminating the above drawbacks and which enables easy performance of all various applications of capillary electrophoresis with one and the same apparatus. In order to achieve this, the invention is characterized by what is set forth in the characterizing clause of claim 1.
In order to perform a given electrophoresis application, it is necessary to choose certain initial and boundary conditions. The regulation of boundary conditions in a capillary electrophoresis system means that the composition of a background solution present in the proximity of the ends of a capillary must be controlled. According to the invention, this is performed by continuously pumping fresh solution past the ends of a separating capillary. This also prevents reaction products resulting from electrode reactions from passing into the capillary. In order to avoid the high consumption of a background solution, the solution channels must have as small a volume as possible. The design or an apparatus according to the present invention is based on this principle. In the apparatus of the invention, the test conditions can be chosen without restrictions and modified arbitrarily during a run.